Over the last few years there has been a tremendous growth in the area of salivary diagnostics, prompted in part by efforts from the National Institutes of Health (NIH) and its National Institute of Dental and Craniofacial Research (NIDCR) division to promote the development of non-invasive technologies for the diagnosis of diseases and measurement of specific analytes or molecules in saliva or oral fluid samples. These agencies have made funding available for the development of novel, innovative tools including microarrays, lab-on-a-chip, lateral flow, ELISA and other technologies using saliva and other non-invasive sampling methodologies. Other reasons for the increased interest in salivary diagnostics include the development of important new technologies for both the collection and testing of oral fluids and improvements in the manufacturability of such devices.
There are several successful corporate stories in salivary diagnostics that are also prompting other companies to search out opportunities in non-invasive testing. For instance, it is now possible to detect HIV antibodies from oral specimens at the point-of-care using the OraQuick® HIV 1/2 rapid antibody test (OraSure Technologies, Bethlehem Pa., USA) with greater than 99% sensitivity and specificity. This company is currently proceeding with an FDA submission that would allow consumers to purchase such a test over the counter in a pharmacy.
In the United States millions of oral specimens are collected and processed in the Public Health and insurance market sectors for HIV antibodies, cotinine (nicotine) for smoking and cocaine as part of a “risk assessment” profile. Additionally, options for testing Federal employees for a range of abused drugs using non-invasive methods including saliva are readily accepted by SAMHSA, the Substance Abuse Mental Health Services Administration (the US Government body responsible for drug testing in the Federal workplace). Such testing is also common in the workplace environment where corporations in the US (and other parts of the world) use saliva as part of pre-employment and random drug testing policies.
General wellness is monitored by testing various steroid hormones in the laboratory. Home collection using a standardized saliva collection kit is common place and a large number of testing laboratories have appeared offering a multitude of test options for the “worried well”. Tests include a range of male and female hormones including testosterone, estradiol, progesterone, cortisol and others. In these situations no “diagnosis” is provided on collected samples. Instead, a Medical Director from the laboratory will provide an indication of the levels of a specific target analyte in the saliva and make recommendations on suitable lifestyle changes or perhaps an imminent visit to the doctor.
The future for saliva testing also holds great promise since the publication of the entire salivary proteome by the Human Saliva Proteome Consortium, a group headed by Dr. David Wong from UCLA. The widespread publication of the identity of 1,166 proteins potentially implicated in disease progression will lead to the rapid growth in new applications for salivary diagnosis. Already in development are a diagnostic device for the rapid detection of the drug phenytoin, which uses a surface plasma resonance imaging instrument, an oral fluid Nanosensor test that measures four oral fluid markers as an indication of oral cancer and a lab-on-a-chip technology for point-of-care detection of salivary biomarkers in periodontitis, among a number of others. An excellent review of these and other new applications for oral fluid diagnostics was published following a landmark meeting of many researchers in the field at Lanier Lakes, Ga. USA in October 2006. The monograph published following the meeting is available from the New York Academy of Sciences.
Tools for oral fluid diagnosis may be categorized as one of two types. The first is diagnostic devices providing an immediate diagnosis or test result, so-called “point-of-care” or “near-patient” tests. This category includes the manual OraQuick® rapid HIV 1/2 antibody test that employs lateral flow immunochromatographic technology and the prototype Oral Fluid Nanosensor (OFNASET) Test device from Dr. David Wong's UCLA laboratory, which relies on microfluidics, nanotechnology in a hand-held reading device to potentially screen for oral cancer. This technology detects a series of four salivary molecular RNA markers. Another example among many others is a rapid point-of-care drug screening technology called RapiScan® from Cozart Biosciences (Abingdon, UK), which is used to screen would be drug offenders at the roadside using saliva samples. Many other rapid diagnostic products exist which require saliva sampling or testing, but such products are outside the scope of this application.
The other side of the market for salivary diagnostics involves the collection of oral fluids and the subsequent transportation of the samples to a laboratory, or other remote site where the testing is performed. Again many examples exist. Examples include the collection of oral samples for HIV testing for Public Health and also for insurance risk assessment, where oral specimens are collected using the OraSure® Oral Fluid Collection Device (OraSure Technologies, Bethlehem Pa., USA) and sent to a laboratory. Typically, specimens are analyzed using traditional ELISA technology for the detection of HIV, cotinine (nicotine), cocaine and others. Others include collection of saliva specimens for drug testing in the workplace environment for pre-employment purposes or random drug testing. In such situations saliva is collected using one of a number of available commercial saliva collection devices (including Intercept™ from OraSure Technologies, Bethlehem Pa., USA, Quanti-SAL™ from Immunalysis Corporation, Pomona Calif., USA, Aware Messenger™ from Calypte Biomedical, Lake Oswego, Oreg. USA and Salivette®, Sarstedt, Germany among others) then sent to a laboratory where a battery of drug tests including marijuana (THC), cocaine, opiates (heroin), methamphetamine, amphetamine, and phencyclidine is tested on the processed saliva. Similar practices are observed in Federal workplace and military drug testing environments.
A small industry has emerged for salivary hormone testing where laboratories provide saliva collection kits and a test menu for home users. Clients expectorate into a tube that is subsequently sent to a laboratory. As part of the service subjects are able to request testing for various steroid hormones as part of a general wellness screening panel. The results provide an indication of general health and wellness, without providing any definitive diagnosis.
Very recently a new industry has emerged for “personal genome” testing in what is termed the “consumer genetics” market. In this area saliva or buccal cell swab samples are collected in the home and sent to a laboratory and tested for specific genetic markers and single nucleotide polymorphisms (SNPs) that provide information on the parentage of the individual in question, predisposition to specific diseases, ancestry and other genetic information. The number of companies in this area is rapidly growing but at this time, the recognized market leading companies are 23andMe, Navigenics, DeCode Genetics, Knome, Illumina, and Sciona. The convenience and non-invasiveness makes saliva very attractive for home testing/home collection products. Other applications in this market sector will be addressed in further detail below.
In general, multi-purpose saliva collection is facilitated using one of a number of commercially available saliva collection devices or by expectoration (“spitting”) into a sample receptacle. A number of devices are now available to collect specimens and these include the OraSure® device (OraSure® Technologies), Aware Messenger™, Salivette, Omni•SAL® (Stat-Sure Diagnostics, Framingham, Mass., USA), ORACOL (Malvern Medical Developments, UK), Cozart Oral Swab (Cozart BioSciences, Abingdon, UK) and the Versi•SAL® device (Oasis Diagnostics® Corporation, Vancouver, Wash. USA). With the exception of the Versi•SAL® device, which provides the opportunity to use multiple absorbent materials, customized to specific applications, these products have limited applications. This is mainly due to limitations in the number and type of absorbent materials used to perform the saliva collection operation. While each of the above methodologies may be considered appropriate for certain applications in salivary testing, none of these devices is appropriate for the collection, stabilization, transportation and extraction of purified DNA from saliva. This in turn has restricted the use of salivary DNA for “downstream” applications particularly the potential use of saliva specimens for molecular diagnostic testing.
Molecular diagnostics is one of the fastest growing areas in the area of clinical and animal diagnostics. The current market for molecular diagnostics is estimated to be $3.2 billion (2007 figures) and forecast to reach $5.4 billion by 2012. In this area of clinical diagnostics traditional blood testing is by far the current method of choice. In current protocols, specimens are collected in a blood tube, usually by a trained phlebotomist, and sent to the laboratory. Upon receipt at the laboratory, the sample is initially separated from unwanted blood by-products then further purified prior to analysis. Blood samples contain potentially infectious agents and the cost of transportation can be expensive. In addition, all samples must be treated as infectious waste and disposed of according to recognized safety standards, which can also be costly. If a device was available to collect salivary DNA for clinical diagnostic testing this would offer several advantages over current blood testing algorithms and would be welcomed in clinical practice as a step forward. From the patient's perspective it would eliminate painful blood draws associated with current testing. In addition it would eliminate the need for a trained phlebotomist to draw the blood sample, as well as alleviate any potential for infection from tainted blood samples. Overall, saliva sampling is generally cheaper and does not require an additional pre-treatment step (as required for blood), to separate the required salivary component prior to analysis.
As described previously, there are a number of commercially available saliva collection devices on the market. In most cases, these devices incorporate some sort of absorbent material that is used to collect the saliva specimen. The sample is subsequently removed from the absorbent material using methods such as squeezing, centrifugation or simply soaking in a buffer to solubilize the target analytes. These devices work well for the collection of certain molecules such as infectious disease antibodies (including HIV, hepatitis B, hepatitis C and others), hormones, cancer biomarkers and drugs, for instance, but none of these may be applied to the collection and retrieval of DNA (Deoxy Ribonucleic Acid) or RNA (Ribo Nucleic Acid), which requires a device with very specific performance characteristics. This is due to an inherent property of current devices to bind DNA and RNA moieties to the fibers of the absorbent material used to collect the specimen. DNA binds tightly to the fibers and is not easily removed. Any effort to remove the DNA, cells using reagents, organic solvents usually results in denaturation of the DNA molecules and subsequently observed recoveries are poor.
Some of the above limitations have been overcome in a few devices that do successfully facilitate salivary DNA collection. Expectoration (spitting in a cup or other vessel) provides a saliva sample that can be successfully stabilized and purified through available methodologies to yield high quality DNA, and this method is in use in various testing strategies, however this method lacks adequate standardization (sample variability) and is not considered elegant or dignified.
Over the last few years other promising devices have emerged that are based upon modifications to the traditional expectoration technique. The most widely used of these is the OraGene® DNA device from DNA Genotek (Ottawa, Ontario, Canada). OraGene® is a more sophisticated way to collect saliva into a vessel to which is attached a screw-on cap. In the screw-on cap is a mixture of preservative buffers. Upon completion of the expectoration process, the cap is screwed onto the device releasing the preservative buffer, which drops into the saliva, is mixed by shaking and then acts to protect the integrity of the sample until processing and extraction can take place. The same company has recently perfected the OraGene® RNA device for the collection of RNA from oral fluid specimens. OraGene® RNA applies the same basic principles as used in the OraGene® DNA device. Invitek Gesellschaft für Biotechnik and Biodesign mbH (Invitek, Berlin, Germany) has come up with a similar tool, SaliGene® as an alternative “spit-in-a-cup” technology, which has additional application as a collector for stool or swab specimens [when coupled with specific extraction kits for these alternate specimen types]. In the SaliGene® device, subjects expectorate into a modified collection tube until a pre-determined volume has been reached. A screw-cap with attached plunger is screwed in place and the plunger depressed causing a preservative/lysis buffer to flow into the collected saliva specimen. The sample of mixed preservatives and saliva is gently shaken then sent to a laboratory for further processing.
Researchers from Roswell Park describe the extraction of genomic DNA from saliva using the Qiagen (Hilden, Germany) QIAamp Kit on the Qiagen website. This work was reprinted from earlier work carried out in 1997. The QIAamp kit is one of a number of kits commercially available for DNA extraction from bodily fluids. In this case as in many others, expectoration was used to collect the saliva specimens.
In collecting specimens for diagnostic testing several criteria are important. Specimens need to be collected rapidly to eliminate any opportunity for sample degradation, and they must be removed rapidly from the point of collection and stabilized promptly for subsequent transportation purposes (if necessary). The specimen device used to collect saliva should be able to withstand temperature fluctuations and the rigors of shipping products by air or road allowing samples to arrive safely at the final destination laboratory, hospital or other remote facility. The sample so obtained should be stable for extended periods of time at ambient temperatures and also at −20 degrees Celsius for long-term storage.
Devices for DNA or RNA sample collection need to be robust, transportable, capable of transporting a saliva sample (or other biological fluid) containing the DNA or RNA molecules to a laboratory or other remote facility and also need to provide easy samples removal for subsequent extraction using a number of commercially available, off the shelf kits. The yield of DNA/RNA produced depends upon the particular application but should be sufficient for immediate application in testing kits provided by a multitude of manufacturers for infectious diseases, oncology, cardiovascular diseases, immunological disorders and many others. Literature reports suggest that a minimum of 10 μg of pure DNA should be collected and typically even larger quantities are required. For example, 100 μg or more of pure DNA would be a preferable sample quantity.
While the limited number of examples of salivary DNA devices described above provides methods for DNA/RNA collection and extraction, none of the above devices meet the market need for a simple, elegant, standardized and rapid method for the collection of biological fluids, with the specific purpose of extraction of DNA and RNA, for large scale implementation.